Regulation of gene expression by sigmaS, a member of the sigma70 family, is responsible for the transcription of a variety of genes in E. coli that are expressed as the cells enter stationary phase or during certain types of starvation and stress, such as acid stress or hyperosmotic stress. A role for sigmaS in the regulation of virulence factors has also been established in Salmonella sp. Salmonella plasmid virulence genes, spvR and spvABCD are carried on large (50 to 100-kb) plasmids in a variety of Salmonella species including S. typhimurium, S. choleraesuis, and S. dublin. Loss of these plasmids, of SpvR, SpvC and SpvD, or of sigmaS, results in loss of virulence in mouse models and the ability of the cells to multiply in the reticuloendothelial system. Expression of the genes increases dramatically as cells enter station phase and is dependent on sigmaS. The current model proposes that accumulation of sigmaS in response to growth phase, stress, or starvation conditions increases transcription of spvR, and sigmaS and SpvR then act together to increase transcription of the spv operon. Although additional regulatory factors often function to modulate the expression of specific sigmaS-dependent genes, the expression of these genes results largely from increased levels of sigmaS in the cell. In E. coli, sigmaS levels are controlled in a variety of ways, including transcription initiation and translational elongation. Some of the major factors that influence sigmaS levels, however, are post-translational events that lead to increased stability of the protein. During exponential growth at 37oC,sigmaS has a half-life of less than 2 min. As the cells enter stationary phase or encounter certain stress conditions, the half-life of sigmaS increases to greater than 30 min. The ClpXP protease is a cytoplasmic, ATP-dependent protease that is responsible for the rapid degradation of sigmaS during exponential growth. This rapid degradation of sigmaS by ClpXP requires RssB (also called SprE), a protein that shares homology with the family of response regulators and appears to specifically modulate the activity of sigmaS as well as its degradation. The heat shock protein DnaK has also been shown to have a positive role in the post-translational control of sigmaS. DnaK appears to be involved in the transduction of at least two signals, heat shock and carbon starvation, that result in reduced sigmaS turnover. In bacteria, sigmaS-RNA polymerase holoenzyme is responsible for gene expression following the transition from exponential growth to stationary phase or in response to certain types of stress. B. burgdorferi contains rpoS (encoding sigmaS) and our preliminary data indicate that expression of rpoS increases as cultures enter stationary phase. We have mapped a transcriptional start site of rpoS from B. burgdorferi to a potential sigma54-dependent promoter. Furthermore, analysis of the transcription of rpoS in a B. burgdorferi sigma54 mutant indicates that sigmaS is regulated by sigma54 as cells enter stationary phase of growth. Thus sigma54-holoenzyme is required for sigmaS expression and both proteins play key roles in the survival of B. burgdorferi in the tick midgut and in survival in mammalian hosts. (3) Regulation of gene expression by sigma54. As indicated above, we have identified a potential sigma54-dependent promoter that is involved in expression of rpoS. Sigma54, encoded by the rpoN gene, as first shown to be required for the expression of genes involved in nitrogen metabolism in enteric bacteria. It has since been shown to be required in various bacteria for the transcription of genes whose products are involved in such diverse functions as hydrogen metabolism, C4-dicarboxylic acid transport, pilin and flagellar biosynthesis, and degradation of aromatic compounds. Unlike other alternative sigma factors, sigma54 does not share homology with the sigma70 family. Moreover, the mechanism by which sigma54-RNA polymerase holoenyzme (sigma54-holoenzyme) initiates transcription differs from that of other forms of RNA polymerase holoenzyme. sigma54-Holoenzyme recognizes promoters that have conserved elements in the ?12 and ?24 regions, having the consensus sequence 5?-TGGCACN4TTTGC(A/T)-3?. The spacing between the conserved GG and GC doublets (underlined in the consensus sequence) is critical, as any changes in the spacing result in failure of sigma54-holoenzyme to recognize the promoter. Sigma54-Holoenzyme binds to the promoter to form a closed promoter complex, but it is unable to initiate transcription in the absence of an activator protein. The activator binds to sites that are usually located 100-200 bp upstream of the transcriptional start site and makes transient contact with sigma54-holoenyzme through DNA looping. Productive interactions between the activator and sigma54-holoenzyme lead to the isomerization of the closed promoter complex to an open complex that is transcriptionally competent. To catalyze this isomerization reaction, the activator must hydrolyze ATP. The mechanism by which the activator couples ATP hydrolysis to open complex formation is not known, but it does not appear to involve phosphorylation of either the activator or sigma54-holoenzyme. The activities of activators of sigma54-holoenzyme are regulated in response to environmental signals. Many of the activators of sigma54-holoenzyme are response regulators in two-component regulatory systems, and phosphorylation of these proteins results in their activation. These response regulators are phosphorylated by their cognate protein histidine kinases in response to an environmental signal. Once phosphorylated, the response regulator activates transcription of other genes. The activator of sigma54-holoenzyme from B. burgdorferi is also a response regulator of a two-component system. The gene encoding the activator is in an operon with a gene encoding its cognate protein histidine kinase. Our preliminary data indicate that the activator controls expression of rpoS. Therefore, we refer to this activator as sigmaS regulator (SisR) and its cognate protein histidine kinase as sigmaS regulatory protein histidine kinase (SisK).